Detection and elimination of adventitious agents in continuous cell lines.

The National Veterinary Services Laboratories (NVSL) routinely monitors continuous cell lines (CCL's) used for veterinary biologicals and diagnostic virology. All veterinary biologicals produced in CCL's must follow the master seed concept which limits the use of the master seed CCL to up to 20 passages beyond the passage level characterized and deposited at NVSL. All CLL's are evaluated for the presence of adventitious agents such as mycoplasma, bovine viral diarrhea virus, and other bacteria and viruses. Previously, CCLs were evaluated for tumorigenicity by the Syrian hamster cheek pouch method; however, this procedure has now been eliminated. The adventitious agents most frequently detected in CCL's have been bovine viral diarrhea virus and mycoplasma. Our laboratory has consistently found that the source of bovine viral diarrhea contamination of CCLs has been the use of contaminated fetal bovine cell culture enrichment serum. Gamma irradiation at 2.5-3.5 megarads at -40 degrees C of carefully screened fetal bovine serum has been used in the Diagnostic Virology Laboratory for over 10 years. If the irradiated serum is used at a final concentration of 10 percent, there is no untoward effect on cell susceptibility for virus propagation or cell culture growth. Gamma irradiation has also been demonstrated to be a very efficient inactivator of mycoplasma. Specific conditions utilized by our laboratory to preserve fetal bovine serum cell culture growth factors while eliminating adventitious bovine viral diarrhea virus will be presented.

Standard antisera produced in ponies for the identification of bovine mycoplasmas: comparative growth-inhibition results from six laboratories.

Antisera to 10 mycoplasma species of bovine origin were produced in 10 ponies and were distributed for evaluation in growth-inhibition tests at 6 laboratories in Australia, England, Denmark, France, and the United States. Except for a few failures with some antigens produced at the 6 laboratories, the antisera induced large zones of growth inhibition in homologous, but not heterologous, systems. These antisera may be useful as standard reagents for the identification of the bovine mycoplasmas.

A comparison of a disposable membrane filtration system with a direct inoculation system for the sterility testing of veterinary biologics.

A direct inoculation method for sterility testing veterinary biologics was compared with a closed membrane filtration method (Steritest). The filtration method detected extraneous contamination in 29% more batches of biologics than the direct inoculation method. Live viral biologics produced in cell culture were found to be the only product type that could be filtered; hence the filtration method cannot be recommended universally as a replacement for the direct inoculation method.

Inactivation of mycoplasma in serum with binary ethyleneimine.

A method of inactivating mycoplasma contaminants in sera by using binary ethyleneimine was tried. When used at the concentration recommended for inactivating viruses, 0.001 M, binary ethyleneimine inactivated only two of five mycoplasma species. Even at a concentration of 0.01 M, the compound did not consistently inactivate all mycoplasmas.

Isolation of porcine parvovirus from commercial trypsin.

The isolation of porcine parvovirus from a lot of commercial 1:250 trypsin is reported and the method is described.

Effect of algicidal quaternaries on the germicidal activity of chlorine on swimming pool water.

The Swimming Pool Water Disinfectant Test Method of the Association of Official Analytical Chemists was used to determine the effect of the accepted level of 2 ppm of some commercial quaternary ammonium algicides on the germicidal activity of chlorine. Accurate determinations on the amounts of residual available chlorine in chlorine-quaternary mixtures could not be made by the usual chemical methods. This made it necessary to base all comparisons on the starting concentrations of available chlorine rather than the final concentration as specified in the method employed. No evidence was obtained to support the use of lower concentrations of residual available chlorine for disinfection in the presence of algicidal quaternaries than those commonly recognized as effective by the American Public Health Association. The rate of kill against the gram-positive test organism Streptococcus faecalis was faster in quaternary-chlorine mixtures than in the sodium hypochlorite control solutions. The practical significance of this result in the bench method identified cannot be ascertained in the absence of more sensitive and precise chemical procedures for determining concentrations of residual available chlorine in the presence of quaternaries or in actual swimming pool tests.

Comparison of chlorine, bromine, iodine as disinfectants for swimming pool water.

Studies on the germicidal activity of chlorine, bromine, and iodine were made by use of the Association of Official Agricultural Chemists official first action method for determining effectiveness of swimming pool water disinfectants. In this procedure, 0.3 ppm of available chlorine as chlorine gas has activity equivalent to 0.6 ppm of available chlorine in the buffered sodium hypochlorite control when Escherichia coli is used as the test organism. With Streptococcus faecalis as the test organism, 0.45 ppm of available chlorine as gaseous chlorine gives activity equivalent to the control. Liquid bromine at 1.0 ppm is as effective as the 0.6 ppm of available chlorine hypochlorite control with E. coli as the test organism, but 2.0 ppm of liquid bromine is necessary to provide activity equivalent to the 0.6 ppm of available chlorine control when S. faecalis is employed. With iodine as metallic iodine, 2.0 ppm is necessary to provide a result equivalent to the 0.6 ppm of available chlorine control with both E. coli and S. faecalis. In the various systems tested, gaseous chlorine was the most active form of available chlorine; liquid bromine provided the most active form of bromine, and metallic iodine provided the most active form of iodine.